Survival of nontypeable Haemophilus influenzae in macrophages.

In this study we have investigated the ability of nonencapsulated, nontypeable Haemophilus influenzae, NT477 to survive in the J774 mouse macrophage-like cell line. Viable, intracellular nontypeable H. influenzae could still be recovered from macrophages 72 h after phagocytosis. In contrast, H. influenzae strain Rd, an avirulent, nonencapsulated variant of a serotype d strain, was killed within 24 h. These differences suggest that NT477, in comparison to Rd, possesses unique attributes that enable it to survive in macrophages for prolonged periods. To determine whether this trait is ubiquitous amongst nontypeable H. influenzae, 33 primary clinical isolates obtained from children with otitis media were screened for their ability to survive in macrophages. Of these isolates, 82% were able to persist in an intracellular environment for periods of at least 24 h. The number of viable organisms recovered at this time ranged from 2x10(4) to 50 colony-forming units per strain indicating that the extent to which nontypeable H. influenzae can resist macrophage-mediated killing varies between strains.

Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium.

Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side-chain with structural homology to the human blood group antigen Lewis X (Le(x)). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side-chain. In contrast, an O antigen side-chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti-Le(x) monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin-embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, Le(x) structures in the O antigen side-chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of Le(x) in this interaction was confirmed by the tropic binding of synthetic Le(x), conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild-type H. pylori.

Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectin-saccharide interactions.

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.

The identification a novel gene required for lipopolysaccharide biosynthesis by Haemophilus influenzae RM7004, using transposon Tn916 mutagenesis.

Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Gal alpha (1-4) beta Gal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Gal alpha (1-4) beta Gal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A, a gene which is also required for biosynthesis and phase variable expression of the Gal alpha (1-4) beta Gal LPS epitope.

Tandem repeats of the tetramer 5'-CAAT-3' present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae.

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemophilus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neisseria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)(n) encoded by multiple tandem repeats of 5'-CAAT-3'. This observation suggested that (SINQ)(n) might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5'-CAAT-3' repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)(n) is not a prerequisite for the enzymatic function of Lic2A and that the 5'-CAAT-3' repeats in lic2A function solely as a mechanism for generating phase variation. This observation suggested that wide variation in the number of 5'-CAAT-3' repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5'-CAAT-3' repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)(n) indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.

Phase variation of Haemophilus influenzae lipopolysaccharide: characterization of lipopolysaccharide from individual colonies.

The lipopolysaccharide (LPS) of Haemophilus influenzae expresses a number of core oligosaccharide epitopes on its outer surface. The expression of individual epitopes is subject to frequent (approximately 1% bacteria/generation) reversible phase variation, as determined by colony immunoblots. We have used a microtechnique for the extraction of LPS from individual colonies, whose LPS antigenic phenotype has been identified, so that the LPS can be studied by tricine sodium dodecylsulphate polyacrylamide gel electrophoresis (T-SDS-PAGE). This avoids the introduction of heterogeneous phase-varying LPS which is inevitable if bacteria from colonies are grown in broth culture prior to LPS extraction and analysis. Using these techniques we have investigated the repertoire of LPS phase variation exhibited by H. influenzae strain RM7004 (a serotype b meningitis isolate). This technique will facilitate the study of bacteria in which there is variable LPS expression.

The role of a repetitive DNA motif (5'-CAAT-3') in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope alpha Gal(1-4)beta Gal.

Haemophilus influenzae lipopolysaccharide (LPS) contains structures, defined by monoclonal antibodies, which undergo phase variation. This investigation reports the nucleotide sequence of lic2A, which is required for the expression of at least three phase-variable LPS epitopes, one of which has the structure alpha Gal(1-4)beta Gal. lic2A contains multiple tandem repeats of the tetramer 5'-CAAT-3'. Previous studies have correlated changes in the number of 5'-CAAT-3' repeats with the phase-variable expression of the alpha Gal(1-4)beta Gal epitope. To obtain direct evidence for this, the 5'-CAAT-3' repeat region from lic2A was amplified directly from immunostained colonies and sequenced. This demonstrated that the variable expression of LPS epitopes, including alpha Gal(1-4)beta Gal, is in part directly dependent upon the number of copies of 5'-CAAT-3' within lic2A.

Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from Escherichia coli.

A non-fimbrial adhesin (NFA-1) from the uropathogenic Escherichia coli strain 827 responsible for agglutination of human erythrocytes was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both express adhesin at 37 degrees C, but not 18 degrees C nor in the presence of 1% glucose. Adhesin purified from the clone formed high molecular weight aggregates which were resolved to the 21 K dalton subunit protein seen in the wild type strain on denaturation. Binding to human kidney cells by the clone and the wild type E. coli, from which the genes were cloned, were compared in an ELISA assay and shown to be the same. The genes for the adhesin were isolated on a 15.5 kilobase BamHI-EcoRI fragment which was subjected to gamma delta mutagenesis. The NFA-1 operon was localised to a 6.5kb region of this fragment.

A block of urovirulence genes encoding multiple fimbriae and hemolysin in Escherichia coli O4:K12:H-.

Cosmid gene libraries were constructed from a uropathogenic isolate of Escherichia coli O4:K12:H- that secretes alpha-hemolysin and produces the F14, F12-rel, F1C, and F13 fimbrial antigens. A series of overlapping clones was generated, and individual cosmid clones were found to express various combinations of fimbriae and hemolysin, suggesting that the genes for these potential virulence factors are closely linked. By using Southern hybridization analysis and restriction endonuclease mapping, it was demonstrated that the cosmid clones carried a nested set of overlapping, cloned, genomic DNA fragments. A comparison of the phenotypic properties of individual cosmid clones and subclones allowed the order of the gene clusters encoding these factors to be deduced. The cloning also revealed the presence of a fifth fimbria that had P-adhesin specificity.